Removal and preparation of the implant-bone specimens

The implants with a neighboring bone were removed en bloc, and the adhesive soft tissues were dissected to investigate the healing status and the bone-implant contact (BIC) percentage. The specimens were fixed in 10% neutral buffered formalin for 48 h and dehydrated in subsequent concentrations of 70–99.9% ethanol. After dehydration, the specimens were embedded in methyl methacrylate (Technovit 7200 VLC, Heraeus Kulzer GmbH & Co. KG, Wehrheim, Germany) without decalcification. 200-μm-thick slides were cut from the blocks using a band saw (Exakt 300 CL, Exakt Apparatebau, Norderstad, Germany).

Histologic and histomorphometric analysis

The 50-μm-thick final histological slides were prepared by grinding with 320–4000 grit sandpapers (Hermes Schleifmittel GmbH & Co. KG, Hamburg, Germany). The final sections were mounted and stained with toluidine blue for histologic and histomorphometric analysis. In order to measure the BIC percentage, digital images of the sections were obtained by a digital camera (Olympus DP 70, Olympus, Tokyo, Japan) attached to a microscope (Olympus BX50). The obtained images were transferred to a computer and were histomorphometrically analyzed using ImageJ analysis software (ImageJ, National Institutes of Health, Bethesda, Maryland, USA) (Fig. 6). The BIC percentage was calculated by an experienced researcher blinded to the study protocol.

Statistical analysis

Statistical analyses were performed using SPSS v.20.0 (IBM, Chicago, IL, USA). The Shapiro-Wilks normality test was used to verify the normality of the data. All variables were normally distributed and thus parametric tests were used for intra-group (paired sample t test) and inter-group (one-way ANOVA/Tukey’s test) comparisons. A p value of < 0.05 was considered significant.